Our focus is on the mechanisms of ventricular fibrillation (VF) and sudden cardiac death (SCO). SCO can occur in young, otherwise healthy individuals suffering from inherited gene mutations. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by adrenergically mediated rounds of bidirectional (biVT) and polymorphic (PVT) ventricular tachycardias, leading to syncope and/or SCO in the absence of structural heart disease; mortality is ~30% by the age of 40 years. As many as 40 point mutations in the human cardiac sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor type 2 [RyR2]), linked to defective SR Ca2+ channel function, have been reported in individuals affected by CPVT and arrhythmogenic right ventricular cardiomyopathy type 2 (ARVC2). Our central objective is to determine the electrophysiological mechanisms of CPVT. We take advantage of a unique knock-in mouse model (RyR2+/RyR2R4496C) that carries the murine equivalent of a human missense mutation (R4497C) in RyR2 that results in CPVT. Recent data show that administration of caffeine and of adrenergic agonists predisposes the RyR2+/RyR2R4496C mouse heart to biVT, PVT and VF, which suggests the involvement of increased Ca2+ release through the defective RyR2 channels. It is our hypothesis that arrhythmias in this model, and by inference in CPVT patients, are triggered by delayed afterdepolarizations (DADs) occurring at Purkinje fibers on the right and left branches of the specialized ventricular conducting system. We will test this hypothesis using an integrative approach from the molecule to the organ level. Our Specific Aims are: 1. To investigate the mechanisms underlying the occurrence of SR-Ca2+ leak in cardiac Purkinje fibers and ventricular myocytes of the RyR2+/RyR2R4496C mouse heart. 2. To determine whether ventricular myocytes and Purkinje cells obtained from RyR2+/RyR2R4496C heart undergo DADs and triggered activity in the presence of increased extracellular Ca2+ or during superfusion with caffeine and/or isoproterenol. 3. To determine the electrophysiological mechanisms of biVT, PVT and VF in the RyR2+/RyR2R4496C mouse heart. The results derived from the proposed studies should provide understanding to the CPVT phenotype and give mechanistic insight in conditions such as heart failure and others in which there is an increased vulnerability to arrhythmias due to abnormal SR Ca2+ release. [unreadable] [unreadable] [unreadable]